Adenovirus vectors have proven highly potent for inducing both humoral and cellular immune responses. The failure of an Ad5-Gag/Pol/Nef vaccine in the Step study has led to the development of novel serotype Ad vectors, which are biologically very different and arguably substantially superior to Ad5 in terms of seroepidemiology, cellular receptor usage, in vivo tropism, innate immune responses, transcriptional profiles, adaptive immune phenotypes, and protective efficacy against SIV in rhesus monkeys. Moreover, the inclusion of Env may be critical for an HIV-1 vaccine. Based on our preclinical and clinical preliminary data, we have prioritized Ad26/MVA and Ad26/Protein regimens for further development. In Project 1, we will conduct the key preclinical studies to inform the clinical development program described in Project 2. These studies will help define the lead vaccine candidate and will help determine the mechanism of protection afforded by our optimal vaccine regimen. Specifically, we will test the hypothesis that adding a gp140 trimer protein boost will augment the partial protection afforded by our optimal vaccine vectors against acquisition of highly stringent, heterologous SIV challenges. We will also test the hypothesis that vaccine-elicited Env-specific antibodies are critical for this protection. To evaluate these hypotheses, we propose the following two Specific Aims: 1. To compare the immunogenicity and protective efficacy of Ad26/Ad26 and Ad26/MVA vector regimens with and without a gp140 trimer protein boost against repetitive, heterologous, neutralization-resistant, intrarectal SIV challenges in rhesus monkeys; and 2. To define the mechanism of blocking acquisition of stringent SIV challenges by conducting antigen formulation and adoptive transfer studies in rhesus monkeys.